Alpha-class glutathione transferases as steroid isomerases and scaffolds for protein redesign

Sammanfattning: The present work focuses on the glutathione transferase (GST) Alpha-class enzymes, their characteristics as steroid isomerases and structural plasticity as malleable scaffolds for protein design. The GSTs are a family of detoxication enzymes that appears to have a wider variety of additional functions.Kinetic steady-state parameters for human GST A1-1 with the steroid isomerase substrate ?5-androstene-3,17-dione (AD), an intermediate in steroid hormone biosynthesis, were determined. It was established that GST A1-1 is a highly efficient steroid isomerase with a 30-fold higher catalytic efficiency, in terms of kcat/Km, than 3?-hydroxysteroid dehydrogenase/?5?4-isomerase, the enzyme regarded as the mammalian ?5?4-isomerase in steroid hormone biosynthesis. Kinetic parameters were also determined for GST A2-2, GST A4-4 and the GST A1-1 mutant Y9F. From the dependency on pH of the kinetic parameters it was established that efficient catalysis requires glutathione (GSH) in its deprotonated form and it is suggested that the GSH-thiolate acts as a base in the catalysis of the ?5?4-3-ketosteroid isomerase reaction.GST A2-2 is a poor catalyst of the steroid isomerase reaction while GST A3-3 is highly efficient. Their catalytic efficiencies (kcat/Km) differ 5000-fold. Stepwise point mutations were performed to GST A2-2 in order to insert the amino acid residues from the active-site of GST A3-3 that distinguishes the two isoenzymes. The result was that GST A2-2 was redesigned to a highly efficient double-bond isomerase with both the catalytic constant (kcat) and catalytic efficiency (kcat/Km) in the same order as for GST A3-3. Furthermore, this was done by only exchanging amino-acid residues with first-sphere interactions, providing empirical proof-of principle for knowledge-based enzyme design.Kinetic studies on GST A1-1 and a T68E mutant of GST A1-1 were also performed with a GSH analog lacking the g-glutamate a-carboxylate (dGSH), and using three different electrophilic substrates (AD; 1-chloro-2,4-dinitrobenzene, CDNB; 4-nitrocinnamaldehyde). Deletion of the a-carboxylate from the GSH glutamate had a severe impact on all reaction constants and it changed the rate-limiting step for the CDNB reaction as well as changed the pKa value for the enzyme-bound GSH thiol. The loss in activity caused by dGSH could in part be compensated by the T68E mutant contributing an enzyme-bound carboxylate instead.The C-terminus of GST A1-1 is flexible and folds over the active site when the enzyme binds a substrate. Phenylalanine residues in the C-terminal end, known to interact with active-site residues tyrosine 9 and phenylalanine 10, were mutated to abolish those interactions. Studies of viscosity dependence for CDNB and AD with regard to kcat and kcat/Km showed that the dynamic C-terminal segment influence rate-determining steps for both the larger isomerase substrate, AD, as well as for the smaller conjugation substrate, CDNB.